I propose a genetic analysis of the mechanism(s) of enzyme processing and localization for enzymes of the lysosome-like vacuole of Saccharomyces cerevisiae. We will extend analysis of the defect in pep4 mutants, which fail to process the precursor of carboxypeptidase Y (CPY) in this organism and which has reduced levels of at least five enzymes of the vacuole. We will search for precursors for the enzyme affected by the pep4 mutations and will continue analysis on amother mutation, pep17-1, which has a similar phenotype. Additional mutants with pleiotropic defects for vacuolar enzymes will be sought and, if found, chracterized. We will attempt to determine where the precursor(s) accumulates in the mutant(s) and will attempt to delineate a pathway for processing and localization. We will clone the PEP4 (and other relevant) gene(s) by isolating DNA plasmids capable of complementing the pep4-3 after DNA-mediated transformation. Once isolated, we plan to engineer plasmids which will produce large amounts of the PEP4 gene product in bacteria after being attached to the lac promoter and/or lac z gene. This will allow us to develop reagents for assaying the quantity and location of the PEP4 gene product in the cells and should lend insight into the role the PEP4 gene has in enzyme processing and/or localization. The project should yield information on the pathways, markers, and/or vehicles for processing and localization and where processing events occur.